With further expansion of our biological atomic force (Bio-AFM) technology, instrumentation and data analysis methods, we have advanced a number of biomedical investigations in collaboration with NIH intramural and extramural researchers. Major sub-projects with notable results include: (1) We have investigated the macromolecular structure of a recombinant Plasmodium falciparum Merozoite Surface Protein 3 (MSP3), a recombinant Plasmodium falciparum circumsporozoite protein (CSP), and two other malaria vaccine candidates via AFM in collaboration with Dr. David Narum (NIAID, NIH) and coworkers at Malaria Vaccine Development Branch lead by Dr. Louis Miller (NIAID, NIH). These protein antigens for malaria vaccine are being produced from Escherichia coli, purified, and characterized in a manner suitable for scale-up toward human trials. We have focused on using AFM and related studies to understand the structural properties of these antigens under a range of solution and perturbative conditions. Our results have implications for protein-protein interactions and human immunological response, and have contributed to two publications and other manuscripts in preparation. (2) We have continued our AFM studies of clathrin and clathrin coated vesicles (CCVs) in collaboration with Dr. Ralph Nossal (NICHD, NIH), Prof. Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio) and coworkers. Clathrin triskelia form the outer clathrin lattice cages of the CCVs during subcellular trafficking via interactions with adaptor proteins, membrane lipids, and other cofactors. The intricacies of these dynamic macromolecular constructs have inspired numerous structural and functional studies. We have resolved variable profiles of triskelia on mica surfaces for the first time by AFM at a resolution comparable to electron microscopy. Classical tri-leg, filamentous pin-wheel shapes, as well as non-planar triskelion conformations and triskelion-triskelion dimers, are readily observed both dried on mica surface and under buffers. Pentagonal and hexagonal lattice structures are well visualized in a variety of clathrin assemblies with or without AP180 adaptors, similar to those of the native CCVs purified from bovine brains. We have also produced single molecule force spectroscopy (SMFS) of triskelia and CCVs under buffers and revealed, also for the first time, a series of internal energetic barriers that characterize triskelion heavy chain folding and unfolding, including molecular sequence and structure periodicity for both the seven repeating 145aa motifs and numerous 30aa hairpins. The dynamic stability of these domains has been obtained. A manuscript on these results is under review and another manuscript is in preparation. (3) We have explored with a number of additional intramural and extramural collaborators to develop and apply further AFM nanotechnology in such areas as structure and function of G-protein coupled receptor (GPCR), mycobacterium tuberculosis membrane, live cells, bacterial bio-films, polymer and biomimicking materials, and fundamental surface sciences.